title: E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display.
abstract: Here we describe a protocol for the selection of full-length IgG antibodies from repertoires displayed on Escherichia coli. In the method described here, full-length heavy and light chains are assembled in the periplasm into aglycosylated IgGs that are fully functional for antigen binding. Expression of an inner membrane-tethered Fc-binding protein is used to capture the IgG molecules and anchor them to the cell. Following outer-membrane permeabilization, fluorescently labeled ligand-binding library clones are selected by multiple rounds of fluorescence-activated cell sorting. Selection of a comprehensive set of IgG clones can typically be obtained within 3-4 weeks, a timescale that is comparable with most prevalent antibody display technologies. The isolated antibodies are well expressed in bacteria and exhibit affinities per binding site in the nanomolar range.
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Yariv
authors.initials: Y
authors.lastname: Mazor
authors.forename: Thomas
authors.initials: T
authors.lastname: Van Blarcom
authors.forename: Brent L
authors.initials: BL
authors.lastname: Iverson
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000302:isolation & purification*; D019151:Peptide Library; D019897:Periplasm / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D019151:Peptide Library; D011993:Recombinant Fusion Proteins